Interpretive Summary: Spread of Rift Valley fever virus from endemic areas to North America is a serious threat to livestock, wildlife, and people. Currently, regional diagnostic labs do not have the ability to diagnose RVFV because all diagnostics and reagents available must be used in high containment labs. In this study, we developed and evaluated two diagnostic assays to detect virus that can be safely run in typical veterinary diagnostic labs. These assays will give our regional diagnostic labs the capacity to diagnose RVFV in the event of an introduction.

Technical Abstract:
Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants with an overall human infection rate of <1%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. A one-step real-time RT-PCR (rRT-PCR) assay was developed where samples are quickly inactivated allowing the assay to be performed in BSL-2 laboratories. The assay was evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 106.5 plaque forming units/ml, or up to 1010 viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, anti-recombinant RVFV- nucleocapsid (N) antibody was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these reagents and assays will be useful to national laboratories as initial diagnostic tests with confirmation by virus isolation. These assays can be safely produced and distributed to regional diagnostic laboratories, thus providing capacity for early detection of RVFV in suspected ruminant samples.